Mechanism of N₂ Fixation and Ammonia Assimilation

Two metalloproteins i.e. larger Mo-Fe-protein and smaller Fe-protein components are involved in N₂ fixation. Fe-protein interacts with ATP and Mg⁺⁺, and receive electron from ferredoxin or flavodoxin when it is oxidized. Mo-Fe-protein of nitrogenase complex combines with the reducible substrates i.e. N₂ and yields two molecules of NH₃. It appears that N₂ is reduced step-wise without breaking N-N bond until the final reduction and production of ammonia is accomplished. Finally two molecules of NH₃ are released from the enzyme (Fig. 11.3).

Mechanism of electron transport is shown in Fig. 11.4. Fe-protein (oxidized form) gets electrons from ferredoxin (when it combines with 2H⁺ and yields H₂) and energy from ATP. Mg⁺⁺ activates this reaction.

Finally, electron is transferred to oxidize Mo-Fe-protein which becomes reduced and Fe-protein is oxidized. It is the reduced form of Mo-Fe-protein which combines with N₂ and other substrates to result in NH₃ and other various products with respect to substrate. H₂ produced during this reaction is further utilized by some microorganisms which possess hydrogenase. Reutilization of H₂ increases nitrogenase activity by protecting it from inhibition of H₂.

Ammonia is further synthesized into a number of metabolic products in microbial cells. However, ammonia is not accumulated in the cell, although a few species may create it; rather it is incorporated into organic forms by combining with an organic acid (a -keto-glutaric acid) to give rise to amino acid e.g. glutamic acid. The ammonia may also combine with organic molecules to yield alanin or glutamine (Alexander, 1977).

It is evident from Fig. 11.5 that glutamine synthetase (GS) catalyses the reaction of glutamic acid plus NH₃ and converts into glutamine, which in turn combines with 2-oxoglutarate and results in two molecules of glutamic acid in the presence of an enzyme, glutamine oxoglutarate amino transferase (GOGAT). Glutamic acid is the source of several metabolic products such as amino acids, nucleotides, proteins, etc. The 2-oxoglutaric acid is produced by combining maltose with CO₂. Also maltose gives rise to glucose-6-phosphate. Its further conversion in different microorganisms differs. In cyanobacteria, glucose-6-phosphate is converted to ribose 5-phosphate with an intermediate product 6-phosphogluconate, and produces H⁺. In bacteria it differs from genus to genus.

In Clostridium pasteurianum, pyruvic acid is produced from glucose-6-phosphate. However, in R. rubrum pyruvic acid supports nitrogenase activity (Ludden and Burris, 1981) releasing ATP.

Thus, nitrogen fixed by microorganisms is released into their surrounding. Sewart (1970) proved the release of N¹⁵ by Nostoc into sand-dune slack soil.